In mammalian cells, the enzyme HMG CoA reductase catalyzes reduction of HMG CoA to mevalonate, a rate-determining step in the synthesis of cholesterol and non-sterol isoprenoids. Reductase is integrated into the ER membrane through an N-terminal domain that contains eight membrane-spanning helices. The C-terminus of reductase projects into the cytosol and exerts catalytic activity. End-products of mevalonate metabolism accelerate ER-associated degradation (ERAD) of reductase as part of a complex feedback system that ensures cholesterol homeostasis. Excess sterols cause binding of the membrane domain of reductase to ER membrane proteins called Insig-1 and Insig-2, resulting in the poly-ubiquitination of reductase. This ubiquitination is obligatory for recognition and delivery of reductase to cytosolic 26S proteasomes for degradation. The reaction has been reconstituted in Drosophila S2 cells by overexpressing the membrane domain of mammalian reductase and Insig-1 or Insig-2. As a model system to study fundamental questions in biology, S2 cells offer a number of advantages. For example, transgenes can be easily overexpressed in S2 cells for study of their function and RNAi is simpler and much more effective in S2 cells than in mammalian cells. To gain further insight into mechanisms for Insig-mediated degradation of reductase, we propose three specific aims: 1) Determine mechanism for sterol-accelerated degradation of mammalian HMG CoA reductase in S2 cells; 2) Define role of Hrd1 ubiquitin ligase complex components in sterol-accelerated degradation of reductase in mammalian cells; and 3) Identify novel genes required for degradation of reductase through a genome-wide RNAi screen in S2 cells. Collectively, these studies will provide crucial information regarding mechanisms for degradation of reductase and other polytopic proteins from the ER. In addition, these studies have significant clinical implications. Reductase is the target of statins, a family of widely prescribed drugs that lower blood LDL-cholesterol and reduce the incidence of coronary artery disease. Statins trigger responses that lead to accumulation of active reductase protein, thereby blunting their effects. Part of this increase is due to slowed degradation of reductase. Thus, elucidating mechanisms for the ERAD of reductase may lead to new therapies that increase the effectiveness of statins and ultimately reduce the incidence of heart attacks. PUBLIC HEALTH RELEVANCE: The key enzyme in cholesterol synthesis is HMG CoA reductase, which is tightly controlled through multiple mechanisms that includes regulation of protein stability. Competitive inhibitors of HMG CoA reductase called statins are routinely used to lower blood cholesterol, but they trigger regulatory responses that lead to the accumulation of reductase protein. This grant will investigate the mechanism for the degradation of reductase, the elucidation of which will provide insight into development of therapies to counteract statin-induced accumulation of reductase and improve the effectiveness of the drugs.